The invention is in the field of label free sensing, including label free electronic biomolecular detection using field effect transistors, including ion-sensitive-field-effect-transistors. The invention generally relates to methods for analyte detection and associated devices, particularly analytes comprising biological material suspended in biological material compatible fluids having high relatively high ionic strength.
There is a need in the art for low-cost reliable biological sensors for detecting various analytes. Although a variety of biosensors are available, many conventional sensors require use of a label to assist with analyte detection, including optical labels such as fluorescent dyes and the like, or require more involved amplification steps to amplify a target of interest, such as by polymerase chain reaction. Such conventional sensing systems suffer a common disadvantage of requiring sample preparation, such as one or more of washing, isolation, incubation, temperature control and other handling dependent on the biosensor type.
Field effect transistors (FET) are useful candidates for biosensors to address the above limitations. FETs can be of extreme sensitivity and rely on change in electrical signals attributed to the presence of analytes of interest, thereby avoiding a need for labels or amplification. Examples of various applications using FETs include U.S. Pub. Nos. 2014/0054651, 2011/0086352 and 2012/0021918; PCT Pub. Nos. WO2013/016486, WO2013/173754, WO2012/078340.
There are, however, certain limitations associated with FET sensing, particularly as applied to biological applications. For example, salt concentration is known to strongly influence the sensitivity of FET-based biosensors. The effect of shielding effect of excess ions, which is often represented by the “Debye length”, on detecting biomolecules is examined by Stern et al. (Importance of the Debye Screening Length on Nanowire Field Effect Transistor Sensors, Nano Lett., vol. 7, no. 11, pp. 3405-3409, November 2007.) Nair and Alam (“Design Considerations of Silicon Nanowire Biosensors,” IEEE Trans. Electron Devices, vol. 54, no. 12, pp. 3400-3408, 2007) analyzed the effect of high salt concentration on the electrical characteristics of nanowire-based FET sensors and show strong links between sensitivity and ionic strength and demonstrate that the response of these sensors rapidly tails off as physiological conditions of 0.15M salt is approached. Further, in a recent review, Rajan et al. (Performance limitations for nanowire/nanoribbon biosensors: Performance limitations for nanowire/nanoribbon biosensors,” Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol., vol. 5, no. 6, pp. 629-645, November 2013.) also discussed both the need to overcome ionic shielding and strategies to achieve the same—either through antibody fragmentation, or biomolecule pre-concentration followed by solution exchange to low ionic sensing buffers.
The use of an electrical method, by applying DC fields between a pair of electrodes, to deplete ions from aqueous solution is used in the field of seawater desalination. Capacitive Deionization (CDI) involves the electrophoretic movement of counterions towards charged electrodes, which are then absorbed within the “Debye capacitance layer” to deplete the bulk salt concentration (T. J. Welgemoed and C. F. Schutte, “Capacitive Deionization Technology™: An alternative desalination solution,” Desalination, vol. 183, no. 1-3, pp. 327-340, November 2005; J.-H. Lee, W.-S. Bae, and J.-H. Choi, “Electrode reactions and adsorption/desorption performance related to the applied potential in a capacitive deionization process,” Desalination, vol. 258, no. 1-3, pp. 159-163, August 2010; O. N. Demirer and C. H. Hidrovo, “Laser-induced fluorescence visualization of ion transport in a pseudo-porous capacitive deionization microstructure,” Microfluid. Nanofluidics, July 2013.) See also U.S. Pat. No. 8,377,280 and Pat. Pub. No. 2010/0140096. A similar electrical method (membrane electrodialysis) for desalting with the use of ultraporous membranes is described in U.S. Pat. No. 6,284,117, for primarily PCR-SDA assay techniques.
A common strategy to address sensitivity of FETs is to perform sensing in low ionic buffers. This involves functionalization as well as sensing at low concentrations, far from physiological conditions, or physical desalting after binding in high salt to low sensing buffer concentrations by fluid swap through multiple washing steps (see Stern et al.; and Kim et al. “Direct label-free electrical immunodetection in human serum using a flow-through-apparatus approach with integrated field-effect transistors,” Biosens. Bioelectron., vol. 25, no. 7, pp. 1767-1773, March 2010.). Other strategies include biomolecule fragmentation and pre-concentration methods.
Alternatively, complex AC excitation strategies have been incorporated to break the electrochemical double layer formation and sustain electric fields beyond the Debye length for sensing larger biomolecules (“Detection beyond the Debye Screening Length in a High-Frequency Nanoelectronic Biosensor,” Nano Lett., vol. 12, no. 2, pp. 719-723, February 2012.). Again this method relies on electrophoretic movement of ions and the use of high-frequency excitations to perturb electric fields in solution at faster speeds than the mobility of ions.
Implementation of on-chip desalting around sensors generally include microfluidic in-line membrane-based desalting of analyte for FET based detection (U.S. Pat. Pub. No. 2009/0142825). Also described are electrophoretic schemes (DC electric fields using electrodes around mixing channels) for microfluidic desalting for protein enrichment in mass spectroscopy sensors (“Advanced cleanup process of the free-flow microfluidic device for protein analysis,” Ultramicroscopy, vol. 108, no. 10, pp. 1365-1370, September 2008).